Our goal is to characterize the function of excitatory amino acids as synaptic transmitters in the vertebrate central nervous system using biophysical, physiological and pharmacological techniques for experiments on dissociated cultures of mouse CNS. Pharmacological experiments in vivo suggest separate receptors selectively activated by kainate, quisqualate and N-methyl-D-aspartate (NMDA). Micromolar concentrations of magnesium ions cause a voltage-dependent block of ion channels linked to NMDA, but not kainate or quisqualate receptors. The block by Mg++ shows relief on extreme hyperpolarization. At mM concentrations calcium also produces a partial block of inward current flow through NMDA receptor ion channels. The blocking action of Mg++ is reduced by Ca++, especially on extreme hyperpolarization, suggesting competition between Ca++ and Mg++ for a site within the ion channel. One interpretation of this would be to suggest that Ca++ ions have a significant permeability through NMDA receptor ion channels. Reversal potential measurements on changing extracellular Ca++ confirm this. Measurement of intracellular Ca++ with the dye, arsenazo III, also shows significant calcium influx through NMDA but not kainate or quisqualate receptor ion channels. In addition to amino acids, small peptides are attracting attention as transmitter candidates. N-acetylaspartylglutamate (NAAG) has been proposed as a transmitter in the olfactory cortex. In our experiments, based on dose response curves, pharmacological antagonism, and noise analysis, NAAG behaved as a weak but selective agonist at NMDA receptors and is unlikely to act as a synaptic transmitter. Synaptic potentials mediated by activation of NMDA receptors have been studied in spinal cord cultures. The NMDA receptor mediated component of the epsp is of long duration and low amplitude, but has the same latency as the epsp mediated by quisqualate or kainate receptors. These experiments suggest that a dual receptor mechanism with distinct conductances may underlie neurotransmission at synapse using excitatory amino acids as transmitters.